Activation of vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone

J. Insect Physiol., 1974, Vol. 20, pp.

1815to 1823.Pvgamon Press. Printed in Gent Britain

ACTIVATION OF VITELLOGENIN THE MOSQUITO AEDES AEGYPTI ANN

MARIE

FALLON,*

H. H. HAGEDORN,? HANS LAUFER

SYNTHESIS IN BY ECDYSONE G. R. WYATT,*

and

Department of Biology, Yale University, New Haven, Connecticut 06520; and Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268, U.S.A. (Received 21 January

1974)

Abstract-Injected t%ecdysone was found to induce the synthesis of yolk protein (vitellogenin) in adult female Aedes aegypti without a blood meal. After injection of 5 pg ecdysone per mosquito, vitellogenin constituted 80 per cent of the total protein secreted by explanted fat body, a proportion comparable to that produced by fat body from blood-fed females. Moreover, the time course of induction of vitellogenin synthesis in ecdysone-injected mosquitoes was similar to that triggered by a blood meal. Response to ecdysone is dosedependent: 0.5 pg per female was required to stimulate synthesis to 50 per cent of the level found 18 hr after a blood meal. Ecdysone was effective in decapitated or ovariectomized mosquitoes, and also when applied directly to fat body preparations in vitro. Thus it appears that ecdysone acts directly on the fat body to induce specific protein synthesis, as does the vitellogenin stimulating hormone (VSH) from the ovary of blood-fed mosquitoes. These results suggest that ecdysone can replace VSH in inducing vitellogenin synthesis in the unfed mosquito.

INTRODUCTION

EGG DEVELOPMENT in insects involves synthesis of yolk proteins, or vitellogenins, by the fat body. These proteins are released into the haemolymph and selectively incorporated into the developing oiicytes (TELFER, 1960; BROOKES,1969; ENGELMANN, 1969; PAN et al., 1969; HAGEDORNand JUDSON, 1972). Different insects have evolved various modes of hormonal regulation of this process (DOANE, 1973). In the mosquito, Aedes aegypti, egg development is triggered by a blood meal which effects the release from the corpus cardiacurn of an egg development neurosecretory hormone (EDNH) produced in the brain (LEA, 1972). The EDNH either directly or indirectly causes the ovary to produce a vitellogenin stimulating hormone (VSH) (HAGEDORN,1974), which activates and maintains vitellogenin synthesis in the fat body (HAGEDORNand FALLON, 1973). * Present address : Department of Biology, Queen’s University, Kingston, Ontario, Canada. t Present address : Department of Entomology, University of Massachusetts, Amherst, Massachusetts 01002, U.S.A. 1815

1816

ANN MARIE FALLON,H. H. HAGEDORN,G. R. WYATT, AND HANS LAUFER

Injection of the insect moulting hormone, /?-ecdysone, can cause the mosquito to produce viable eggs without a blood meal (SPIELMAN et al., 1971). This suggested to US that the synthesis of vitellogenin by the fat body might be activated by ecdysone. The nature of the response of the fat body to ecdysone is the subject of this paper. MATERIALS

AND METHODS

Mosquitoes Aedes aegypti of the Rockefeller strain were reared in the laboratory by standard techniques (JUDSON, 1967) at 24°C with an 8 hr dark period. Adults were used to 4 10 days after emergence and maintained at 27°C during the experiments. Operations Adult mosquitoes were ligated by tying a silk thread around the neck, followed by decapitation. Ovaries were removed through slits between the fifth and sixth abdominal segments: Sham operations involved a similar wounding without ovariectomy. Mosquitoes were allowed to recover for 24 hr before injection. Injections /%Ecdysone (of plant origin, prepared by Takeda Pharmaceutical Industries, Japan) was kindly given by Dr. H. CHINO (Hokkaido University); analysis by gas-liquid chromatography carried out by Dr N. IKEKAWA (Tokyo Institute of Technology, Tokyo, Japan) showed about 3% contamination with inokosterone, Inokosterone is a closely related steroid known and no other detectable impurities. to share biological activity with ecdysone (see WYATT, 1972). The ecdysone was dissolved in 50% ethanol and diluted with Aedes saline (VARMA and PUDNEY, 1969) to a final ethanol concentration of 5% for injection. Saline containing 5% ethanol was used for controls. For the experiments in vitro, ecdysone was dissolved directly in medium, without ethanol. Tests in vitro, performed after the injection experiments had been completed, showed an inhibitory effect of 5% ethanol. For injection, chilled insects were immobilized on a surface under netting, and 1~1 of liquid was injected between the thoracic pleura with a micromanipulator and a needle drawn from a glass capillary. Treated animals were kept in containers lined with moist cotton and covered with parafilm to prevent dehydration. Vitellogenin synthesis in organ culture The assay for vitellogenin synthesis was essentially that of HAGEDORN et al. (1973). The mosquito abdomen was isolated and its last two segments were pulled away, removing the ovaries, gut, and Malpighian tubules. The abdomen was split on one side, leaving the fat body attached to the body wall, and floated on culture medium with the fat body hanging freely in the liquid. Except in the time course experiment, vitellogenin synthesis was assayed 18 hr after injection. The medium contained the salts and glucose mixture described by VARMA and PUDNEY (1969) and the amino acid mixture used for Cecropia fat body organ

VITELLOGENIN SYNTHESIS IN AEDES ACTIVATED BYECMONSE

1817

culture (REDDY and WYATT, 1967). Polyvinylpyrrolidone (2%) (JUNGREISand WYATT, 1972) was included in the medium when subsequent trichloroacetic acid (TCA) precipitation was not required. Fat bodies from 10 females were incubated in 0.10 ml of medium containing 5 &i 3H-phenylalanine (Amersham Searle, Inc.) in plastic tubes shaken gently in a water-bath at 25°C. After 3 hr, the medium was removed, and the tissue rinsed twice with 0.050 ml of saline. The medium and washings were combined and centrifuged for 5 min in a Beckman microfuge. The fat bodies were frozen for subsequent protein determination. To determine vitellogenin synthesis, 0.025 ml portions of the centrifuged medium were added to O-025 ml of appropriately diluted antiserum to purified yolk protein, and then 0.005 ml of carrier yolk protein (1 mg/ml) was added. The tubes were incubated in a 37°C water-bath for 1 hr, and then kept on ice for an hour or more (HOROWITZand SCHARFF,1969). The precipitate was collected on 0.45 pm Millipore filters presoaked in 0.1 M sodium phosphate buffer, pH 7.2, containing 0.9% NaCl and approximately O*l”,b DL-phenylalanine, 0*30/, bovine serum albumin, and 0.3% polyvinylpyrrolidone. The filters were washed with 40 ml of phosphate-buffered saline, followed by 2 ml chloroform-methanol (1 : l), dried, and counted in toluene-based scintillation fluid. To isolate total protein in the medium, 0.050 ml of 10% TCA was added to 0.050 ml portions of medium. The mixture was kept in the cold overnight, and the precipitate was collected on Millipore filters presoaked in 50/, TCA containing phenylalanine and rinsed with 40 ml of 5% TCA. To determine total fat body protein, fat bodies were thawed, sonicated in O-20 ml of 0.05 M Tris-Cl buffer, pH 8.0, containing O-25 M NaCl, and centrifuged. Protein was determined in 0.025 ml samples by the method of BRAMHALL et al. (1969), and was used as a basis for expressing the incorporation of label into vitellogenin or total protein secreted into the medium. The data therefore do not represent specific activities.

RESULTS To establish that the yolk deposition reported by SPIELMAN et al. (1971) represented production of vitellogenin and not non-specific mobilization of nutrient reserves we first injected non-blood-fed (unfed) mosquitoes with 5 and 10 pg doses of ecdysone. The latter dose gave a maximum response in SPIELMAN’S work. Ecdysone injection stimulated synthesis of the specific protein, both 5 and 10 pg giving essentially the same response (Table 1). In the fed mosquito, vitellogenin constitutes 85 to 90 per cent of the protein secreted into the medium by the fat body (HAGEDORNand JUDSON,1972). Induction of synthesis by injected ecdysone results in a similar ratio of vitellogenin to total protein (Table 2). To determine the effective dose of ecdysone, we injected quantities ranging from 0.005 to 10 pg per female. A sharp rise in vitellogenin synthesis occurred between 0.05 and 0.5 pg (Fig. 1). S’mce the mean wet weight of a mosquito is

1818

ANN MARIE FALLON,H. H. HAGEDORN,G. R. WYATT, ANDHANS LALJFER TABLE ~-EFFECT OF INJECTED

ECDYSONE

Fed females Saline injected Unfed females Saline injected Ecdysone injected 5 Pg 1OcLg

ON

VITELLOGENIN

SYNTHESIS

n

Counts/min in antibody ppt per pg fat body protein f S.E.

4

65.3 f 5.5

4

5.6fl.O

3 3

50.5 f 2.8 51.4+ 1.5

Data represent averages from several independent experiments errors. Fat bodies from 10 females were used in each replicate.

!z standard

TABLE ~-SPECIFICITY OF THE EFFECTOF ECDYSONE ON VITELLOGENIN SYNTHESIS

Fed females Saline injected Unfed females Ecdysone injected 5 tLg 1OcLg

Counts/min in antibody ppt per pg fat body protein

Counts/min in TCA ppt per pg fat body protein

Ratio Antibody/TCA

64.3

74.9

0.86

50.9 52.9

63.0 67.3

0.81 0.79

Each value represents the mean counts/min females each. Average deviation = 5 per cent.

from duplicate samples of 10

2.5 mg, this dose range corresponds to about 0.02 to 0.2 mg ecdysone per g wet weight. A single injection of ecdysone resulted in a sharp rise in vitellogenin synthesis which continued for 24 hr and then declined (Fig. 2). This curve might reflect the biological inactivation of the hormone and, if so, a second dose should restore a high rate of synthesis. Synthesis was not restored, however, by a second injection (Fig. 2). It seemed possible that ecdysone might be acting on the fat body through some other hormone. To test this we injected ecdysone into neck-ligated and decapitated females. Synthesis occurred, indicating that ecdysone could not be exerting its effects by way of the brain (Table 3). In addition, ecdysone stimulated vitellogenin synthesis in ovariectomized mosquitoes (Table 3), which shows that its action was independent of the ovary. Therefore, the evidence was consistent with a direct action of ecdysone on the fat body. The reduced response to ecdysone after ligature

VITELLOGENIN SYNTHESISIN AEDES ACTIVATED BY ECDYSONE

2 go-

‘=

0 0

2-

so-

v

zs 70?j 8 60a? v;5ci ==. 40u) .E 305 g zo0) C '

IO0'

I

0005

I

0.05

I

I

0.5

Ecdysonedose /Indtvidual,

I

50 IX pg

FIG. 1. Vitellogenin synthesis in response to varying amounts of ecdysone. The data are averages of three independent experiments, each including 2 replicates of 10 fat bodies each. The control value (65.3 counts/min per pg) was from females injected with saline immediately after feeding and assayed 18 hr later.

Time ofter

ecdysone injection,

hr

2. Time course of response to ecdysone. Mosquitoes were injected with 5.0 pg of ecdysone per female, and vitellogenin synthesis was assayed at the times indicated. 0, Rate of synthesis 24 hr after a second injection given at the time indicated by the arrow. Each point represents the mean from triplicate samples of 10 fat bodies each + the standard error. FIG.

1819

1820

ANN

MARIE FALLON, H. H. HAGEDORN,G. R. WYATT, ANDHANS LAUFER

or ovariectomy panying these

(compare Table manipulations.

TABLE 3-EFFECT

3 with Table

1) may be due to the injury

accom-

OF INJECTEDECDYSONEON OVARIECT~MIZED ANDNECK-LIGATED MOSQC’ITOES

Fed females Sham operated, Saline injected Unfed females, neck ligated Saline injected Ecdysone injected 0.5 CLg 5.0 Pg Unfed females Sham operated, Saline injected Ecdysone injected 0.5 CLg Ovariectomized, Ecdysone injected 0.5 Pg 5.0 CLg

Tl

Counts/min in antibody ppt per pg fat body protein t S.E.

6

53.5 + 6.2

3 3 3

3

1.8 rt 0.13 7.3 + 0.4 20.0 f 1.3

2.0 + 0.26

3

22.8 t 1.7

3 3

12.5 + 1.3 23.7 + 2.8

Values represent averages from several independent experiments errors. Fat bodies from 10 females were used for each replicate. TABLE ~-INDUCTION OF VITELLOGENIN SYNTHESISIN FAT BODY FROM BY ECDYSONEDURING INCUBATIONin vitro

+ standard

UNFED

MOSQUITOES

Counts/min in antibody ppt per pg fat body protein Experiment Unfed females 1. Incubation 12 hr with ecdysone and isotope present throughout 2. Incubation 21 hr with ecdysone present throughout and isotope during last 3 hr only Fed females 3. Incubation 3 hr, 18 hr after feeding

- ecdysone

+ ecdysone

I.0

18.4

3.8

96.6

148.8

-

Ten fat bodies were incubated in 0.1 ml of medium containing 5 &i 3Hphenylalanine and (when present) 50 pg ecdysone. Values are means from two replicates.

VITELLOGENIN SYNTHESIS IN AEDES ACTIVATED BYECDYSONE

1821

The induction of vitellogenin synthesis in fat body preparations from unfed females in vitro (Table 4) further supports the hypothesis of a direct action of ecdysone on the fat body. After an 18 hr incubation in the presence of ecdysone, the rate of synthesis was similar to that of the fed mosquito 18 hr after the blood meal. In these experiments, ecdysone was added at O-5 mg/ml; lower levels, which might have been effective, were not tested.

DISCUSSION We

have shown that ecdysone can act on the fat body of an adult insect, selectively stimulating synthesis of vitellogenic protein. Ecdysone is effective in ovariectomized or decapitated mosquitoes, and also when applied in vitro to fat body preparations from adult females. Thus, neither the ovaries nor any extra-abdominal endocrine organ is required for the stimulation of vitellogenin synthesis by ecdysone. It is likely then, that ecdysone is acting directly on the fat body, although we have not excluded a possible role of other tissues included in the fat body preparation, such as oenocytes. Both the vitellogenin stimulating hormone (VSH) from the ovary (HAGEDORNand FALLON, 1973; HAGEDORN,1974) and ecdysone act independently of the brain and corpus allatum. Thus it seems reasonable that ecdysone acts by replacing VSH. The time course of the ecdysone response indicates that the induction and cessation of vitellogenin synthesis is similar to that in the blood-fed animal (HAGEDORNet al., 1973). A second ecdysone injection at the peak of vitellogenin synthesis failed to restimulate or prolong high rates of synthesis. Thus, there appears to be a block to further synthesis of vitellogenin after 24 hr. These observations are consistent with evidence showing that the developing ovary produces a second humoral factor that prevents yolk deposition in the penultimate follicles (MEOLA and LEA, 1972; ELSE and JUDSON,1972). It is possible that this inhibitory factor from the ovary acts on the fat body to arrest vitellogenin synthesis. Is ecdysone identical to VSH ? The ecdysial glands, generally regarded as the principal source of ecdysone, degenerate when insects metamorphose to the adult stage (see BURGESSand REMPEL, 1966, for mosquitoes). Thus it is commonly believed that adult insects lack ecdysone. However, ecdysone has been identified in some adult insects-locusts (STAMM, 1958), the silkmoth Bombyx (4-8 x 10-s g per g wet wight) (SHAAYAand KARLSON, 1965), and milkweed bugs (3 x 10-s g per g wet weight) (FEIR and WINKLER, 1959). Although these are levels at which ecdysone is active in many insect systems, they are far below the amount that we have found to be effective in inducing vitellogenin synthesis after injection. Possibly, however, the injected hormone is subject to rapid inactivation. A possible role of ecdysone in relation to oogenesis is suggested by indirect evidence for relatively high titers of ecdysone in female adults of some other species. BODENSTEIN (1943) f ound that imaginal disks implanted into adult female Drosophila grew, whereas the contralateral disks implanted into males did not grow unless ring glands were also implanted. Even in the presence of ring glands, disks grew better

1822

ANNMARIEFALLON,H. H. HAGEDORN, G. R. WYATT, ANDHANS LAUFER

in females than males. Similar results have been found in the mosquito Culex pipiens and other insects (see GEHRINGand NOTHIGER, 1973). Furthermore, in adult Bombyx, females apparently have a higher ecdysone titre than males (SHAAYA and KARLSON,1965). Since the completion of these experiments, SCHLAEGER et al. (1974) have found fi-ecdysone in adult female A. uegypti after a blood meal at levels reaching 3 x lo-’ g per g wet weight. This further suggests that ecdysone may have a natural r81e in vitellogenin synthesis in mosquitoes. These considerations lead us to conclude that ecdysone may well be identical to the ovarian VSH, although production of ecdysone by insect ovaries has never been reported. A more confident interpretation of the r8le of ecdysone in this system awaits determination of its source in adult mosquitoes. Acknowledgements-This work was supported by grants from the Whitehall Foundation, the National Institutes of Health (HD-02176), the National Science Foundation (GB 18606), and the Connecticut Research Foundation. We thank Drs. H. CHINO, T. OHTAKI, and N. IKEKAWAfor the gift and analysis of a sample of p-ecdysone, and Dr. M. S. Fuchs for informing us of his findings before publication. REFERENCES BODENSTEIND. (1943) Hormones and tissue competence in the development of Drosophila. Biol. Bull., Woods Hole 84, 34-59. BRAMHALLS., NOACKN., WV M., and LOEWENBERCJ. R. (1969) A simple calorimetric method for determination of protein. Analyt. Biochem. 31, 146-148. BROOKED V. J. (1969) The induction of yolk protein synthesis in the fat body of an insect, Leucophaea maderae, by an analog of the juvenile hormone. Devel. Biol. 20,459-471. BURGESSL. and REMPEL S. G. (1966) The stomodaeal nervous system, the neurosecretory system, and the gland complex in Aedes aegypti. Can. J. 2001. 44, 731-765. DOANEW. W. (1973) Role of hormones in insect development. In Developmental Systems: Insects (Ed. by COUNCES. J. and WADDINGTONC. H.) 2, 291497. Academic Press, New York. ELSE J. G. and JUDSONC. L. (1972) Enforced egg-retention and its effects on vitellogenesis in the mosquito, Aedes aegypti. J. med. Ent. 9, 527-530. ENGELMANNF. (1969) Female specific protein: biosynthesis controlled by corpus allatum in Leucophaea maderae. Science, Wash. 165, 407-409. FEIR D. and WINKLER G. (1969) Ecdysone titres in the last larva and adult stages of the milkweed bug J. Insect Physiol. 15, 899-904. GEHRINGW. J. and NOTHIGERR. (1973) The imaginal discs of Drosophila. In Developmental Systems: Insects (Ed. by COUNCES. J. and WADDINGTON C. H.) 2, 212-290. Academic Press, New York. HAGEDORNH. H. (1974) The control of vitellogenin synthesis in the mosquito. Am. 2001. In preparation. HAGEDORN H. H. and FALLONA. M. (1973) Ovarian control of vitellogenin synthesis by the fat body in Aedes aegypti. Nature, Lond. 244, 103-105. HAGEDORNH. H., FALLONA. M., and LAUFERH. (1973) Vitellogenin synthesis by the fat body of Aedes aegypti: evidence for transcriptional control. Devel. Biol. 31, 285-294. HAGEDORN H. H. and JUDSONC. L. (1972) Purification and synthesis of Aedes aegypti yolk proteins. J. exp. 2001. 182, 367-377. HOROWITZM. S. and SCHARFFM. D. (1969) Fundamental Techniques in Virology (Ed. by HABEL K. and SALZMANN. P.), pp. 297-315. Academic Press, New York.

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